ITAF45 is a pervasive trans-acting factor for picornavirus Type II IRES elements

Abstract

Viruses have evolved elaborate mechanisms to hijack the host mRNA translation machinery to direct viral protein synthesis. Picornaviruses, whose RNA genome lacks a cap structure, inhibit cap-dependent mRNA translation, and utilize an internal ribosome entry site (IRES) in the RNA 5' untranslated region to recruit the 40S ribosomal subunit. IRES activity is stimulated by a set of host proteins termed IRES trans-acting factors (ITAFs). The cellular protein ITAF₄₅ (also known as PA2G4 or EBP1) was documented as an essential ITAF for foot-and-mouth disease virus (FMDV), with no apparent role in cell-free systems for encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelitis virus (TMEV), which are closely related viruses harboring similar IRES elements. Here, we demonstrate that ITAF₄₅ is a pervasive host factor for picornaviruses containing a Type II IRES. CRISPR/Cas9 knockout of ITAF₄₅ in several human cell lines conferred resistance to infection with FMDV, EMCV, TMEV, and equine rhinitis A virus (ERAV). We show that ITAF₄₅ enhances initiation of translation on Type II IRESs in cell line models. This is mediated by the C-terminal lysine-rich region of ITAF₄₅ known to enable binding to viral RNA. These findings challenge previous reports of a restricted role for ITAF₄₅ in FMDV infection, thus positioning ITAF₄₅ as a potential antiviral target for various animal viruses and emerging human cardioviruses.

Keywords: host factor; picornavirus; translation.

Fig. 1.

ITAF₄₅ is a key host factor for EMCV infection. (A) Immunoblot of control, ITAF₄₅^KO, and ITAF₄₅^KO + MYC-DDK-tagged ITAF₄₅ H1-HeLa cells. (B) Crystal violet staining of viable cells infected with indicated virus with “mock” representing uninfected cells. (Also B): Categorization and taxonomy of viruses used and the type of IRES they contain in their 5′-UTR.

Fig. 2.

Depletion of ITAF₄₅ restricts EMCV infection. (A) RT-qPCR quantification of EMCV RNA in control, ITAF₄₅^KO, and ITAF₄₅^KO + ITAF₄₅ infected with EMCV (Mengo strain) at a multiplicity of infection (MOI) of 10 for 7 h. (B). Plaque assay quantification of infectious EMCV produced from control, ITAF₄₅^KO, and ITAF₄₅^KO + ITAF₄₅ cells infected with EMCV (Mengo strain) at an MOI of 10 for 24 h. Datasets represent means ± s.d. (n = 3 independent biological replicates). All P values were determined by ordinary one-way ANOVA using GraphPad Prism (GraphPad Software) with Dunnett’s correction. ***P < 0.0002 and ****P < 0.0001.

Fig. 3.

ITAF₄₅ stimulates the translation of EMCV RNA in human cell lines. (A) Top: Schematic of the full-length EMCV (Mengo strain) genome containing Gaussia luciferase downstream of the 5′-UTR followed by a 3C^pro cleavage site (EMCV-GLuc). Bottom: EMCV-GLuc expression levels in control, ITAF₄₅^KO, and ITAF₄₅^KO + ITAF₄₅ cells transfected with 1 µg of EMCV-GLuc RNA. (B) Top: Schematic of the capped and polyadenylated EMCV IRES bicistronic reporter mRNA used in which the EMCV IRES is between a firefly luciferase cistron (cap-dependent translation) and a Renilla luciferase cistron (IRES-mediated translation). Bottom: firefly and Renilla luciferase expression levels in relative light units (RLU) in control, ITAF₄₅^KO, and ITAF₄₅^KO + ITAF₄₅ cells transfected with 400 ng of EMCV bicistronic reporter mRNA. Cells were harvested at 6 h posttransfection. Background signals from nontransfected cells were subtracted from the measurements. (C) Control and ITAF₄₅^KO cells infected with EMCV-GLuc virus at an MOI of 20 in the presence of 0.1% DMSO or 125 µM cycloheximide (CHX). Datasets represent means ± s.d (n = 3 independent biological). All P values were determined by two-way ANOVA using GraphPad Prism (GraphPad Software) with Dunnett’s correction. ***P < 0.0002 and ****P < 0.0001.

Fig. 4.

ITAF₄₅ is required for viruses containing a Type II IRES. (A) Left: eHAP1 control and ITAF₄₅^KO cells were infected with FMDV (O1 Kaufbeuren) and cultured in an Incucyte to monitor the rate of cytopathic effect (CPE) development via measurement of cell confluency. The average confluency of three biological replicates was plotted against time using GraphPad Prism (GraphPad Software). Right: Representative image of cell monolayers at 12 h postinfection. (B) Replication of FMDV in eHAP1 control and ITAF₄₅^KO cells was assessed via transfection of 90 ng of an FMDV subgenomic replicon wherein the capsid protein coding regions were replaced with a GFP reporter. Replication was assessed using the Incucyte S3 2019B ver2 software to quantify the total integrated green intensity per well using a cutoff value of 0.5. (C) eHAP1 control and ITAF₄₅^KO cells were infected with TMEV (GDVII strain) at an MOI of 50 and lysed at 24 h postinfection to quantify viral RNA using RT-qPCR. (D) Top: eHAP1 control and ITAF₄₅^KO cells were used to titrate ERAV stock where the end-point titers were calculated using the Spearman–Kärber method. Bottom: Crystal violet staining of cells inoculated with indicated virus. TMEV and ERAV dataset represent means ± s.d (n = 3 independent biological replicates). P values were determined by a paired T-test using GraphPad Prism (GraphPad Software) ***P < 0.0002 and *P < 0.0332.

Fig. 5.

The C-terminal lysine-rich region of ITAF₄₅ is required for efficient EMCV infection. (A) Left: Schematic of ITAF₄₅ constructs stably expressed in H1-HeLa ITAF₄₅^KO cells containing C-terminal MYC-DDK tags. Right: Immunoblot of H1-HeLa control, ITAF₄₅^KO, and ITAF₄₅^KO + MYC-DDK-tagged construct cell lines. (B) Crystal violet staining of viable cells infected with EMCV (Mengo strain) at an MOI of 10 for 24 h with “mock” representing uninfected cells.